Alice:Exome/2012-5-24
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Prepare mouse and human genomic DNA from Xu Lab for Exome Capture(using Agilent's SureSelect kit)[edit]
- sheared the samples at biogem
- 5.3.2012 batch --- mouse SCNT samples
sample ID | concentration of gDNA extracted from cell pellets (ng/ul) | ul of gDNA used for shearing (2.5ug each) | H2O volume (ul) | N2 Index used) |
SCNT B7 P2 | 104.5 | 24 | 26 | N2 index 1 |
SCNT B8 P2 | 129.7 | 20 | 30 | N2 index 2 |
SCNT B9 P2 | 162.6 | 15 | 35 | N2 index 3 |
SCNT B10 P2 | 153.8 | 16 | 34 | N2 index 4 |
SCNT B11 P2 | 147.5 | 17 | 33 | N2 index 5 |
SCNT B12 P2 | 149.2 | 17 | 33 | N2 index 6 |
SCNT B13 P2 | 152.9 | 16 | 34 | N2 index 7 |
B14 P2 | 206.2 | 12 | 38 | - |
B6-P1-1 | 161.4 | 15 | 35 | N2 index 8 |
2A-4F-1 | 251.9 | 10 | 40 | N2 index 9 |
2A-4F-33 | 192.1 | 13 | 37 | N2 index 10 |
- 5.18.2012 batch --- Human iPSC samples
sample ID | concentration of gDNA extracted from cell pellets (ng/ul) | ul of gDNA used for shearing (4ug each) | H2O volume (ul) | N2 Index used) |
Y2 | 823.2 | 5 | 45 | N2 index 17 |
Y7 | 967.6 | 5 | 45 | N2 index 18 |
Y8 | 495 | 9 | 41 | N2 index 19 |
Y9 | 482.1 | 9 | 41 | N2 index 20 |
Y16 | 526.8 | 9 | 41 | N2 index 21 |
A4 | 454.8 | 9 | 41 | N2 index 22 (not included in sequencing) |
A6 | 482.2 | 9 | 41 | N2 index 23 |
A20 | 972.9 | 5 | 45 | N2 index 24 |
B3 | 337.9 | 12 | 38 | N2 index 25 |
B18 | 270.1 | 15 | 35 | N2 index 26 |
1EDKD #3 P7 | 304 | 14 | 36 | N2 index 27 |
2EDKD#1 P7 | 210.1 | 20 | 30 | N2 index 28 (not included in sequencing) |
2EDKD#3 P7 | 164.2 | 25 | 35 | N2 index 29 |
PGP1F hiPS 12.5 1x #1 P5 1/2 of 1 6well | 83.9 | 48 | 2 | N2 index 30 |
PGP1F hiPS 12.5 2x #6 P5 1/2 of 1 6well | 109.4 | 37 | 13 | N2 index 31 |
Hues 9 | 378 | 11 | 39 | N2 index 32 |
- N2 index 22 (not included in sequencing due to low yield)
- N2 index 28 (exome capture done, but not included in sequencing; because Blue determined it is not integration free in the last minute)
- NEBNext® DNA Library Prep Master Mix Set for Illumina is used (E6040S)
End-repair Reactions (done on 5-20-2012)[edit]
Fragmented DNA 50 ul 10X End Repair Reaction Buffer 6 ul End Repair Enzyme Mix 5 ul Keep the tube at ~20°C for 30 minutes. Ampure bead purification (1.6x) and elute in 42.5ul ddH2O Note: for blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
A-Tailing reactions[edit]
Blunt-end DNA 43 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul Klenow Fragment (3’-5’ exo-) 5 ul Incubated at 37C for 30min Ampure bead purification (1.8x) and elute in 32.5ul ddH2O
Adaptor ligation[edit]
Prepare 30uM adaptors: 100uM PE-t: 30ul 100uM PE-b: 30ul 10x stoffel buffer: 10ul H2O: 30ul 94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec.
commonly used adaptors: Blunt-end adaptors: 5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH Solexa_1_up 3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH Solexa_1_lo_nop TA adaptors (for the one adaptor protocol): 5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH PE_t_adapter 3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos t_adaptor_rc_s regular Y adaptor: PE_t_adaptor(top) ACACTCTTTCCCTACACGACGCTCTTCCGATC*T 3'-Phosphorothioate bond PE_b_adaptor(bottom) \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-phosphorylation Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
5x Ligation buffer 10ul DNA ligase 5ul DNA adaptor (30uM) 5ul A tailed DNA 30ul Incubate at room temperature for 30 minutes purify the product with Ampure beads (1X) and elute in 42ul ddH2O
PCR amplification[edit]
Ligation products 10ul 10uM PCR_F 1ul 10uM PCR_R_index 1ul (designed by Dinh: [Link] 2X Phusion HF MasterMix 25ul H2O 13ul 4 well for each sample PCR program: 98 °C 30sec -> 7 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. purify the products with Qiaquick columns and elute in 100ul total per sample For index 1-10, take out 50ul from each sample to do another bead purification (1x) for size selection purpose and elute in 42ul ddH2O
- check the amplicon with PAGE before bead purification index 1-10
File:ZhangLab 2 2012-05-08 14hr 28min.jpg
- check the amplicon with PAGE after bead purification index 1-10
File:ZhangLab 2 2012-05-09 16hr 16min.jpg
- check the amplicon with PAGE before bead purification index 17-32
File:ZhangLab 2 2012-05-22 12hr 14min.jpg File:ZhangLab 2 2012-05-22 12hr 16min.jpg
- check the amplicon with PAGE after bead purification index 17-32
File:ZhangLab 2 2012-05-22 21hr 24min.jpg File:ZhangLab 2 2012-05-22 21hr 21min.jpg
library hybridization (done on 5/22/2012)[edit]
- concentrate the samples at RT down to 3.4ul. If the samples dried up completely, resuspend in 3.4ul ddH2O per reaction
- mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
- For every reaction, add 25ul of SureSelect Hyb #1, 1ul of SureSelect Hyb #2 (red cap), 10ul of SureSelect Hyb #3 (yellow cap), 13ul of SureSelect Hyb #4, total is 49ul
- For every 3 reactions, add 75ul of SureSelect Hyb #1, 3ul of SureSelect Hyb #2 (red cap), 30ul of SureSelect Hyb #3 (yellow cap), 39ul of SureSelect Hyb #4, total is 147ul
- prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
- add 5ul of SureSelect Oligo Capture library
- prepare 1:3 dilution of the RNase Block (purple cap), combine 1ul RNase Block with 3ul ddH2O
- add 2ul of diluted RNase Block to each capture library and mix thoroughly by pipetting
- keep tubes on ice
- prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul):
- put 3.4ul of prepped library(800ng) into tube B add 2.5ul of SureSelect Indexing Block #1, 2.5ul of SureSelect Block #2, and 0.6ul of SureSelect Indexing Block #3
- mix well by pipetting and cap the tubes
- heat up the prepped libraries + blockers at 95C 5min and 65C hold
- maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries)
- incubate both tubes at 65C for at least 5 min before go on
- put the SureSelect Oligo Capture Library from step 3.4 into tube C and incubate at 2 min (now the thermocycler should have three tubes per sample incubating at 65C)
- while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
- while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
- mix well by pipetting and the hybridization mix should be around 27-29ul
- add 20ul of mineral oil to each sample
- incubate the hybridization mixture at 65C for 24 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr)
Prepare magnetic beads[edit]
- prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
- resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
- for each hybridization, add 50ul dynalbead to 1.5ml tube
- wash the beads:
- add 200ul of SureSelect Binding Buffer
- mix the beads on a vortex for 5 sec
- put the tubes into a magnetic separator, remove and discard the supernatant
- repeat the above steps (4.1-4.3) for a total of 3 washes
- resuspend the beads in 200ul of SureSelect Binding buffer
Select Hybrid capture with SureSelect[edit]
- estimate the volume of hybridization mix that remained after 24-72hr incubation
- add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
- incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
- briefly spin in a centrifuge
- separate the beads and buffer on a magnetic separator and remove the supernatant
- Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
- incubate the samples for 15 min at RT
- wash the beads:
- separate the beads and buffer on magnetic separator and remove the supernatant
- resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
- incubate the samples for 10min at 65C
- invert the tube to mix as the beads may be settled
- repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
- mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
- incubate the samples for 10 min at RT
- separate the beads and buffer on Dynal magnetic separator
- transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
- Desalt the capture solution with AMPure Bead (1.2x) and elute in 43 ul ddH2O total (combining two capture reactions per each sample set)
post-hybridization amplification and purification[edit]
- Prepare the Post-Capture PCR Master Mix in a 1.5ml tube.
- Aliquot 20μl of captured DNA as template into one PCR tubes/wells. Mix well by pipetting up and down 5 times. 2 well for each sample set
post-capture DNA: 20ul Phusion High-Fidelity PCR Master Mix (2x): 50μl PCR grade water: 26ul Syb_FP5, 100 μM (Final Conc.: 2μM): 2ul Syb_RP7, 100 μM (Final Conc.: 2μM): 2ul Total : 100ul 98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C Qiaquick column purification and elute in 50ul ddH2O total per sample
- PAGE validation:
File:ZhangLab 2 2012-05-21 21hr 21min.jpg File:ZhangLab 2 2012-05-24 09hr 04min.jpg File:ZhangLab 2 2012-05-24 09hr 03min-B3-hues9.jpg